EP3856778A1 - Système et procédé de développement d'anticorps bispécifiques cd30 pour l'immunothérapie de malignités cd30+ - Google Patents

Système et procédé de développement d'anticorps bispécifiques cd30 pour l'immunothérapie de malignités cd30+

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Publication number
EP3856778A1
EP3856778A1 EP19783844.4A EP19783844A EP3856778A1 EP 3856778 A1 EP3856778 A1 EP 3856778A1 EP 19783844 A EP19783844 A EP 19783844A EP 3856778 A1 EP3856778 A1 EP 3856778A1
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EP
European Patent Office
Prior art keywords
seq
region
sequence
similarity
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP19783844.4A
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German (de)
English (en)
Inventor
Jeffrey A. Medin
Lawrence G. Lum
Robyn A.A. OLDHAM
Archana THAKUR
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Medical College of Wisconsin
University of Virginia Patent Foundation
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Medical College of Wisconsin
University of Virginia Patent Foundation
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Application filed by Medical College of Wisconsin, University of Virginia Patent Foundation filed Critical Medical College of Wisconsin
Publication of EP3856778A1 publication Critical patent/EP3856778A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39583—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials not provided for elsewhere, e.g. haptens, coenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the field of the invention is novel bispecific antibodies specific to human CD30, and the use thereof.
  • Bispecific antibodies include antibodies or antibody-like molecules that contain two different binding specificities by combining two different binding moieties. BiAbs have been studied to be used as immunotherapy for tumors in order to increase the immune response against tumor antigens and thus tumor cells.
  • CD30 cell surface molecule is a member of the tumor necrosis factor receptor (TNF-R) superfamily and a transmembrane glycoprotein preferentially expressed by activated lymphoid cells.
  • TNF-R tumor necrosis factor receptor
  • This family of molecules has variable homology among its members and includes nerve growth factor receptor (NGFR), CDl20(a), CDl20(b), CD27, CD40 and CD95.
  • NGFR nerve growth factor receptor
  • CDl20(a) CDl20(b)
  • CD27 CD40
  • CD95 CD95
  • CD30 was originally identified by the monoclonal antibody Ki-l, which is reactive with antigens expressed on Hodgkin and Reed-Stemberg cells of Hodgkin's disease (Schwab et al, Nature 299:65 (1982)). CD30 has been used as a clinical marker for Hodgkin's lymphoma and related hematological malignancies (Froese et al , J. Immunol. 139:2081 (1987); Carde et al, Eur. J. Cancer 26:474 (1990)). It has since been found on a number of hematologic malignancies.
  • CD30 in tumor cells renders it an important target for antibody mediated therapy to specifically target therapeutic agents against CD30-positive neoplastic cells (Chaiarle, R., et al. Clin. Immunol. 90(2): 157-164 (1999)).
  • Hodgkin Lymphoma is often treatable, with 86% surviving over 5 years. However, about 30% of patients relapse, a subset of which develop resistant HL. Refractory or relapsed chemo-resistant disease is more challenging to treat: the 5-year survival rate for these patients is just 31%. CD30 is also expressed in a substantial subset of patients with acute myeloid leukemia (AML) which accounts for 1.2% of all cancer cases in the United States and has a 5-year survival rate of just 26.6%. Relapse following initial therapy is common, and patients who relapse after a stem cell transplantation are typically non-responsive to further therapeutic intervention.
  • AML acute myeloid leukemia
  • the present invention addresses the aforementioned drawbacks by providing isolated bispecific antibodies able to bind CD30 antibodies and a T-cell or NK cell surface antigen for example CD-3.
  • a particular embodiment is a bispecific antibody comprising an anti-CD30 and an anti-CD3 antibody or antigen binding fragment thereof covalently linked.
  • the bispecific antibodies described herein can be used for methods of treating patient populations having CD30+ cancers.
  • the present disclosure provides an isolated bispecific antibody capable of binding human CD30 and to a T cell surface antigen comprising a CD30 antibody or antigen binding portion thereof and a T cell surface antigen antibody or antigen binding portion thereof,
  • the present disclosure provides an isolated bispecific antibody capable of binding human CD30 and to a T cell surface antigen comprising a CD30 antibody or antigen binding portion thereof and a T cell surface antigen antibody or antigen binding portion thereof, the CD30 antibody or antigen binding portion thereof comprising
  • a light chain variable domain comprising a CDRL1 region of SEQ ID NO:2 or a sequence with 85% similarity to SEQ ID NO:2, a CDRL2 region of SEQ ID NO:3 or a sequence with 85% similarity to SEQ ID NO:3, and a CDRL3 region of SEQ ID NO:4 or a sequence with 85% similarity to SEQ ID NO:4 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:6 or a sequence with 85% similarity to SEQ ID NO:6, a CDRH2 region of SEQ ID NO:7 or a sequence with 85% similarity to SEQ ID NO:7, and a CDRH3 region of SEQ ID NO: 8 or a sequence with 85% similarity to SEQ ID NO: 8;
  • a light chain variable domain comprising a CDRL1 region of SEQ ID NO: 10 or a sequence with 85% similarity to SEQ ID NO: 10, a CDRL2 region of SEQ ID NO: 11 or a sequence with 85% similarity to SEQ ID NO: 11, and a CDRL3 region of SEQ ID NO: 12 or a sequence with 85% similarity to SEQ ID NO: 12 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO: 14 or a sequence with 85% similarity to SEQ ID NO: l4a CDRH2 region of SEQ ID NO: 15 or a sequence with 85% similarity to SEQ ID NO: 15, and a CDRH3 region of SEQ ID NO: 16 or a sequence with 85% similarity to SEQ ID NO: 16,
  • a light chain variable domain comprising a CDRL1 region of SEQ ID NO: 18, a CDRL2 region of SEQ ID NO: 19, and a CDRL3 region of SEQ ID NO:20 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:22, a CDRH2 region of SEQ ID NO:23, and a CDRH3 region of SEQ ID NO:24,
  • a light chain variable domain comprising a CDRL1 region of SEQ ID NO: 26, a CDRL2 region of SEQ ID NO: 27, and a CDRL3 region of SEQ ID NO: 28 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:30, a CDRH2 region of SEQ ID NO: 31, and a CDRH3 region of SEQ ID NO:32, or
  • a light chain variable domain comprising a CDRL1 region of SEQ ID NO:34, a CDRL2 region of SEQ ID NO:35, and a CDRL3 region of SEQ ID NO:36 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:38, a CDRH2 region of SEQ ID NO:39, and a CDRH3 region of SEQ ID NO:40.
  • the T cell surface antigen is CD3.
  • the present invention provides an isolated bispecific antibody capable of binding human CD30 and to a NK cell surface antigen, the bispecific antibody comprising a CD30 antibody or antigen binding portion thereof and a NK cell surface antigen antibody or antigen binding portion thereof, the CD30 antibody or antigen binding portion thereof comprising
  • a light chain variable domain comprising a CDRL1 region of SEQ ID NO:2 or a sequence with 85% similarity to SEQ ID NO:2, a CDRL2 region of SEQ ID NO:3 or a sequence with 85% similarity to SEQ ID NO:3, and a CDRL3 region of SEQ ID NO:4 or a sequence with 85% similarity to SEQ ID NO:4 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:6 or a sequence with 85% similarity to SEQ ID NO:6, a CDRH2 region of SEQ ID NO:7 or a sequence with 85% similarity to SEQ ID NO:7, and a CDRH3 region of SEQ ID NO: 8 or a sequence with 85% similarity to SEQ ID NO: 8;
  • a light chain variable domain comprising a CDRL1 region of SEQ ID NO: 10 or a sequence with 85% similarity to SEQ ID NO: 10, a CDRL2 region of SEQ ID NO: 11 or a sequence with 85% similarity to SEQ ID NO: 11, and a CDRL3 region of SEQ ID NO: 12 or a sequence with 85% similarity to SEQ ID NO: 12 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO: 14 or a sequence with 85% similarity to SEQ ID NO: l4a CDRH2 region of SEQ ID NO: 15 or a sequence with 85% similarity to SEQ ID NO: 15, and a CDRH3 region of SEQ ID NO: 16 or a sequence with 85% similarity to SEQ ID NO: 16,
  • the NK cell surface antigen is CD 16.
  • compositions comprising the bispecific antibody described herein.
  • the present disclosure provides a method of treating a patient having a CD30+ cancer, the method comprising
  • the present disclosure provides a method of inhibiting growth of a tumor cell expressing CD30, comprising contacting the tumor cell with an effective amount of the bispecific antibody described herein such that the growth of the cell is inhibited.
  • FIG. 1 depict the specific binding of novel CD30 antibody clones to CD30-
  • CD30+ cells Flow cytometry analysis was preformed using purified antibody on CD30+ cell lines (SU-DHL-1, RPMI6666, 562) and a CD30- cell line (OCIAML2). A commercially available a-huCD30 antibody (clone BY88) and an isotype control were also tested.
  • FIG. 2 depicts specific binding of the CD30 antibodies using flow cytometry on non-transduced Raji (low levels of endogenous CD 30 expression) and LV transduced CD30+ Raji cells (high levels of CD30 expression).
  • FIG. 3 A depicts the percent identity between the light chains of the novel CD30 antibodies to each other and the known CD30 antibody AC 10.
  • FIG. 3B depicts the percent identity between the heavy chains of the novel CD30 antibodies to each other and the known CD30 antibody AC10.
  • FIG. 4 depicts the binding of the antibodies to CD30 as measured by ELISA.
  • FIG. 5 depicts the percent inhibition of AC10 binding to CD30 + cells (SU-DHL-
  • FIG. 6 depicts the percent inhibition of 8D10 binding to CD30 + cells (SU-DHL- 1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.
  • FIG. 7 depicts the percent inhibition of 10C2 binding to CD30 + cells (SU-DHL- 1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.
  • FIG. 8 depicts the percent inhibition of 12B1 binding to CD30 + cells (SU-DHL- 1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.
  • FIG. 9 depicts the percent inhibition of 13H1 binding to CD30 + cells (SU-DHL- 1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.
  • FIG. 10 depicts the percent inhibition of 15B8 binding to CD30 + cells (SU- DHL-l cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.
  • FIG. 11 is a set of bar graphs summarizing the blocking ability of the antibodies to AC10 (left) and 8D10 (right) binding to CD30 + cells.
  • FIG. 12 is a set of bar graphs summarizing the blocking ability of the antibodies to 10C2 (left) and 12B1 (right) binding to CD30 + cells.
  • FIG. 13 is a set of bar graphs summarizing the blocking ability of the antibodies to 13H1 (left) and 15B8 (right) binding to CD30 + cells.
  • FIG. 14A-E shows further characterization of the bispecific antibodies.
  • A A representative drawing of the anatomy of one embodiment of bispecific antibodies of the present invention.
  • B shows flow cytometry analysis of biAb binding to CD3+ or CD30+ cells.
  • C Demonstrates the conjugation assay design.
  • D Summary of results from the conjugation assay.
  • E Representative flow cytometry plots from the conjugation assay showing a mixture of biAb-armed T cells and CD30+ tumour analysed by flow. Panel 1 is unarmed T cells, panel 2 is 8D10 biAb-armed T cells, and panel 3 is 10C2 biAb-armed T cells.
  • FIG. 14 F is an example method of conjugating the CD30+ antibody and CD3 antibody of the present invention.
  • FIG. 15 shows the expression of CD30 on tumor cells.
  • FIGS. 16A-16B are bar graphs depicting cytokines produced by biAb-armed T cells. 8D10 biAb or 10C2 biAb-armed T cells produce the pro-inflammatory cytokines IL-2 (A) and IFN-g (B) when co-cultured with CD30+ tumor cells, indicating that binding of T cells to the biAb is sufficient to trigger T cell activation and cytotoxicity. Armed T cells cocultured with CD30- cells have much lower cytokine production demonstrating the specificity of this approach.
  • FIGS. 17A-17D demonstrate cytotoxicity of the biAbs of the present invention.
  • Chromium release assay demonstrating the cytotoxicity of 8D10 and 10C2 biAb-armed T cells against CD30+ tumor cells.
  • CD30+ tumor cell lines SU-DHL-l (A), RPMI6666 (B) and Raji LV30 (D, Raji cells transduced with CD30 lentivirus) are killed with varying efficacy by 8D10 and 10C2 biAb-armed T cells.
  • Non-transduced Raji cells C, CD30-
  • Activated T cells may express low levels of CD30, which could be detrimental to the success of this biAb therapy.
  • FIGS. 18A-18B demonstrate CD30 negative control cell (A) are not killed by biAb-armed T cells and that the CD30 expression on activated T cells is low enough to avoid elimination by the 8D10 and 10C2 biAb armed T cells (B). Experiment was done similar to FIG. 17.
  • FIG. 19 demonstrates surface palsmon resonance (SPR) analysis of CD30 mAbs using two approaches.
  • A CD30 protein was immobilized on a CM5 chip, and the antibodies were flowed as an analyte to assess affinity.
  • B 8D10, 10C2, or AC10 antibody was immobilized on a Protein G chip, and CD30 protein was flowed as an analyte to assess affinity.
  • the present invention provides isolated bispecific antibodies that can specifically/selectively bind to CD30+ cells and to T cells or NK cells simultaneously. Specifically, in one embodiment, a bispecific antibody that binds to CD30+ and the T cell surface protein CD3 is provided. The present invention also provides in some embodiments methods of treating a subject having a CD30+ tumor using the bispecific antibodies described more herein. In another embodiment, the present disclosure provides a method of inhibiting growth of a tumor cell by administering an effective amount of the bispecific antibodies described herein.
  • the present disclosure provides a method of enhancing a T cell-mediated immune response against a CD30+ tumor cell, the method comprising administering a therapeutically effective amount of the bispecific antibody described herein to increase the T cell-mediated immune response as compared to treatment without the bispecific antibody.
  • bispecific antibody as used herein means an antibody, such as a recombinant antibody capable of specifically and selectively recognizing and binding two different antigens.
  • the antibody can be produced by methods known in the art, including chemical linkage or cell fusion methods.
  • the bispecific antibody is able to bind (a) CD30 and (b) a T cell or NK cell surface antigen.
  • the bispecific antibody is able to bind (a) CD30 and (b) CD3.
  • the bispecific antibody comprises (a) a CD30 binding antibody or antigen binding fragment thereof and (b) a T cell surface antigen biding antibody or antigen binding fragment thereof.
  • both the CD30 and T cell surface antigen binding antibodies or antigen binding fragments are two different antibodies that are covalently or non-covalently linked to each other.
  • the bispecific antibody is a CD30 antibody covalently linked to a CD3 antibody.
  • antibody or “antibody molecule” are used herein interchangeably and refer to immunoglobulin molecules or other molecules which comprise an antigen binding domain.
  • the term “antibody” or “antibody molecule” as used herein is thus intended to include whole antibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibody, and antigen-binding fragments, genetically engineered antibodies, among others, as long as the characteristic properties (e.g., ability to bind CD30) are retained.
  • whole antibodies e.g., IgG, IgA, IgE, IgM, or IgD
  • monoclonal antibodies e.g., chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibody, and antigen-binding fragments, genetically engineered antibodies, among others, as
  • antibody fragment as used herein is intended to include any appropriate antibody fragment that displays antigen binding function, for example, Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimers thereof and bispecific antibody fragments.
  • the bispecific antibodies described herein may be two antibodies that are covalently or non-covalently linked to produce a single bispecific antibody. The two antibodies may be linked by a linker.
  • the term “antibody” includes “antibody fragments” or “antibody-derived fragments” and “antigen binding fragments” which comprise an antigen binding domain.
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al. , Science 242, 423-426 (1988) and Huston et al. , PNAS USA 85, 5879-5883 (1988)) .
  • single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context.
  • Antibodies can be genetically engineered from the CDRs and monoclonal antibody sequences described herein into antibodies and antibody fragments by using conventional techniques such as, for example, synthesis by recombinant techniques or chemical synthesis. Techniques for producing antibody fragments are well known and described in the art.
  • standard molecular biological techniques can be used to transfer the DNA sequences encoding the antibody's CDR(s) to (1) full IgG scaffold of human or other species; (2) a scFv scaffold of human or other species, or (3) other specialty vectors. If the CDR(s) have been transferred to anew scaffold all of the previous modifications described can also be performed. For example, one could consult Biotechnol Genet Eng Rev, 2013, 29: 175-86 for a review of useful methods.
  • the bispecific antibodies disclosed in the present invention may be modified to be humanized antibodies which include the constant region from a human germline immunoglobulin sequences.
  • the term "recombinant human antibody” or “humanized antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as an SP2-0, NS0 or CHO cell (like CHO Kl) or from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies or polypeptides expressed using a recombinant expression vector transfected into a host cell.
  • Such recombinant human antibodies have variable and in some embodiments, constant regions derived from human germline immunoglobulin sequences in a rearranged form.
  • a humanized antibody may comprise the constant regions derived from the human germline immunoglobulin sequence and the "framework" (FR) variable domain residues which are the variable domain residues other than the hypervariable regions (CDRs).
  • the framework of the variable domain usually consists of four FR domains (between the three CDRs, e.g., FR1, FR2, FR3 and FR4) for both the heavy and light chain (e.g., for light chain region would contain: FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4). Therefore, a humanized antibody may have the constant regions and framework from a human immunoglobulin and the CDRs or hypervariable regions from the mouse monoclonal antibodies described herein.
  • fragment refers to fragments of biological relevance (functional fragment), e.g., fragments which can contribute to or enable antigen binding, e.g., form part or all of the antigen binding site or can contribute to the prevention of the antigen interacting with its natural ligands.
  • Fragments in some embodiments comprise a heavy chain variable region (VH domain) and light chain variable region (VL) of the invention.
  • the fragments comprise one or more of the heavy chain complementarity determining regions (CDRHs) of the antibodies or of the VH domains, and one or more of the light chain complementarity determining regions (CDRLs), or VL domains to form the antigen binding site.
  • CDRHs heavy chain complementarity determining regions
  • CDRLs light chain complementarity determining regions
  • CDRs complementarity determining regions
  • variable domains on two different polypeptide chains, heavy and light chain
  • CDRs for each antigen binding site that can collectively come into contact with the antigen.
  • a single whole antibody molecule has two antigen binding sites and therefore contains twelve CDRs.
  • Sixty CDRs can be found on a pentameric IgM molecule.
  • CDR1 and CDR2 may be found in the variable (V) region of a polypeptide chain
  • CDR3 includes some of V, all of diversity (D, heavy chains only) and joining (J) regions. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions.
  • CDR3 shows the greatest variability as it is encoded by a recombination of VJ in the case of a light chain region and VDJ in the case of heavy chain regions.
  • the tertiary structure of an antibody is important to analyze and design new antibodies.
  • proteins and “polypeptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and“polypeptide” refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • Protein and“polypeptide” are often used in reference to relatively large polypeptides, whereas the term“peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide and “polypeptide” are used interchangeably herein when referring to an encoded gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • the antibodies of the present invention are polypeptides, as well the antigen-binding fragments and fragments thereof.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of a single amino acid composition that specifically binds to a single epitope of the antigen.
  • chimeric antibody refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques.
  • Other forms of “chimeric antibodies” are those in which the class or subclass has been modified or changed from that of the original antibody.
  • Such “chimeric” antibodies are also referred to as "class-switched antibodies.”
  • Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art.
  • the present disclosure provides a CD30-antibody covalently linked to a CD3 antibody.
  • CD30 antibodies or antigen binding fragments used in the bispecific antibodies are CD30 antibodies or antigen binding fragments used in the bispecific antibodies.
  • the portion of the bispecific antibody that specifically and selectively binds to CD30 may be an antibody, e.g., monoclonal antibody or antigen binding fragment thereof.
  • the CD30 binding antibody or antigen binding fragment thereof is capable of selectively binding to human CD30, and having a different binding specificity to CD30 than the known anti-CD30 antibody AC10 (monoclonal antibody in brentuximab).
  • AC10 monoclonal antibody in brentuximab
  • binding we mean an antibody capable of binding human CD30 but does not bind to other CD molecules or other cell surface proteins.
  • binding we mean that the antibodies are capable of detection at a given tissue's extracellular membrane by standard methods (e.g., tissue section immunofluorescence assays or flow cytometry).
  • the CD30 binding portion of the bispecific antibody is a monoclonal antibody (MAbs) that target CD30 and derivatives thereof.
  • Suitable monoclonal antibodies include, but are not limited to, monoclonal antibodies 8D10, 10C2, 12B1, 13H1, and 15B8 produced from hybridoma cell lines as described herein.
  • the monoclonal antibodies used in the bispecific antibodies described herein are able to specifically and selectively bind to CD30 (as demonstrated in Figure 1).
  • the CD30 binding antibody or antigen binding fragment thereof capable of binding human CD30 comprising, consisting or consisting essentially of: (a) a light chain variable domain comprising a CDRL1 region of SEQ ID NO:2or a sequence with at least 85% similarity to SEQ ID NO:2, a CDRL2 region of SEQ ID NO:3 or a sequence with at least 85% similarity to SEQ ID NO:3, and a CDRL3 region of SEQ ID NO:4 or a sequence with at least 85% similarity to SEQ ID NO:4 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:6 or a sequence with at least 85% similarity to SEQ ID NO:6, a CDRH2 region of SEQ ID NO:7 or a sequence with at least 85% similarity to SEQ ID NO:7, and a CDRH3 region of SEQ ID NO: 8 or a sequence with at least 85% similarity to SEQ ID NO:8; (b) a light chain variable domain comprising
  • the CD30 binding antibody or antigen binding fragment thereof capable of binding human CD30 comprising, consisting or consisting essentially of: a heavy and a light chain, wherein the antigen binding domain formed by the heavy and light chain is able to bind specifically to human CD30.
  • the anti-CD30 antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a light chain comprising SEQ ID NO: 1 or a sequence with at least 85% similarity to SEQ ID NO: 1, and a heavy chain comprising SEQ ID NO:5 or a sequence with at least 85% similarity to SEQ ID NO:5.
  • the isolated anti-CD30 antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a light chain comprising SEQ ID NO:9 or a sequence with at least 85% similarity to SEQ ID NO:9, and a heavy chain comprising SEQ ID NO: 13 or a sequence with at least 85% similarity to SEQ ID NO: 13.
  • the isolated anti-CD30 antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a light chain comprising SEQ ID NO: 17 or a sequence with at least 85% similarity to SEQ ID NO: 17, and a heavy chain comprising SEQ ID NO:2l or a sequence with at least 85% similarity to SEQ ID NO:2l.
  • the isolated anti-CD30 or antigen-binding fragment thereof comprises, consists essentially of or consists of a light chain comprising SEQ ID NO:25 or a sequence with at least 85% similarity to SEQ ID NO:25, and a heavy chain comprising SEQ ID NO:29 or a sequence with at least 85% similarity to SEQ ID NO:29; and
  • the isolated anti-CD30 antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a light chain comprising SEQ ID NO:33 or a sequence with at least 85% similarity to SEQ ID NO:33, and a heavy chain comprising SEQ ID NO:37 or a sequence with at least 85% similarity to SEQ ID NO:37.
  • the CD30 binding antibody or antigen binding fragment thereof capable of binding human CD30 comprising, consisting or consisting essentially of: (a) a light chain variable domain comprising a CDRL1 region of SEQ ID NO:2, a CDRL2 region of SEQ ID NO:3, and a CDRL3 region of SEQ ID NO:4 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:6, a CDRH2 region of SEQ ID NO:7, and a CDRH3 region of SEQ ID NO:8; (b) a light chain variable domain comprising a CDRL1 region of SEQ ID NO: 10, a CDRL2 region of SEQ ID NO: 11, and a CDRL3 region of SEQ ID NO: 12 and a heavy chain variable domain comprising a CDRH1 region of SEQ ID NO: 14, a CDRH2 region of SEQ ID NO: 15 and a CDRH3 region of SEQ ID NO: 16, (c) a light chain variable domain comprising a CDRL1 region
  • the anti-CD30 antibody is monoclonal antibody 8D10. In another embodiment, the anti-CD30 antibody is monoclonal antibody 10C2. In another embodiment, the anti-CD30 antibody is monoclonal antibody 12B1. In another embodiment, the anti-CD30 antibody is monoclonal antibody 13H1. In another embodiment, the anti-CD30 antibody is monoclonal antibody 15B8. In a preferred embodiment, the anti-CD30 antibody is monoclonal antibody 8D10 or 10C2.
  • the anti-CD30 antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a humanized antibody, a single chain variable fragment (scFv) antibody, a single domain antibody, an antigen-binding fragment and a chimeric antibody.
  • a monoclonal antibody a humanized antibody
  • a single chain variable fragment (scFv) antibody a single domain antibody
  • an antigen-binding fragment and a chimeric antibody.
  • the antibodies comprise a light and a heavy chain that have substantial identity to the polypeptide sequences found in SEQ ID NOs: 1 and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively, or substantial identity in the CDR regions within the heavy and light chain of the antibody or antigen-binding fragment thereof as described herein.
  • the antibodies have at least 85% identity to the light chain and heavy chain found in SEQ ID NOs: l and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively, alternatively at least 90% sequence identity to the light chain and heavy chain found in SEQ ID NOs: l and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively, alternatively at least 95% sequence identity to the light chain and heavy chain found in SEQ ID NOs: l and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively, alternatively at least 97% sequence identity to the light chain and heavy chain found in SEQ ID NOs: 1 and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively, alternatively at least 98% sequence identity to the light chain and heavy chain found in SEQ ID NOs: l and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively, alternatively at least 100% sequence identity to the light chain and heavy chain found in SEQ ID NOs: l and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively.
  • the antibodies have at least 85% identity to the CDR domains described herein, alternatively at least 90% sequence identity, alternatively at least 95% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity, alternatively at least 100% sequence identity.
  • the antibody or antigen binding fragment thereof has at least 85-100% sequence identity within CDRH1, CDRH2 and CDRH3 within SEQ ID NO:5 (e g., SEQ ID Nos.
  • SEQ ID NO: 13 e.g., SEQ ID Nos: 14-16
  • SEQ ID NO:2l e.g., SEQ ID Nos: 22-24
  • SEQ ID NO:29 e.g., SEQ ID Nos: 30-32
  • SEQ ID NO:37 e.g., SEQ ID NOS: 38-40
  • SEQ ID NO:9 e.g., SEQ ID Nos: 10-12
  • SEQ ID NO: 17 e.g., SEQ ID NOs. 18-20
  • SEQ ID NO:25 e.g., SEQ ID Nos: 26-28
  • SEQ ID NO:33 e.g, SEQ ID Nos: 34-36
  • the antibody or antigen binding fragment thereof has at least 95-100% sequence identity within CDRH1, CDRH2 and CDRH3 within SEQ ID NO:5 (e.g., SEQ ID Nos. 6-8), SEQ ID NO: 13 (e.g, SEQ ID Nos: 14-16), SEQ ID NO:2l (e.g, SEQ ID Nos: 22-24), SEQ ID NO:29 (e.g, SEQ ID Nos: 30-32), or SEQ ID NO:37 (e.g., SEQ ID NOS: 38-40) and/or at least 95%-l00% sequence identity within CDRL1, CDRL2 and CDRL3 within SEQ ID NO: l (e.g. SEQ ID Nos.
  • SEQ ID NO:9 e.g., SEQ ID Nos: 10-12
  • SEQ ID NO: l7 e.g., SEQ ID NOs. 18-20
  • SEQ ID NO:25 e.g., SEQ ID Nos: 26-28
  • SEQ ID NO:33 e.g., SEQ ID Nos: 34-36
  • the antibody or antigen binding fragment thereof has 100% sequence identity within CDRH1, CDRH2 and CDRH3 within SEQ ID NO:5 (e.g, SEQ ID Nos. 6-8), SEQ ID NO: 13 (e.g., SEQ ID Nos: 14-16), SEQ ID NO:2l (e.g., SEQ ID Nos: 22- 24), SEQ ID NO:29 (e.g., SEQ ID Nos: 30-32), or SEQ ID NO:37 (e.g., SEQ ID NOS: 38-40) and/or 100% sequence identity within CDRL1, CDRL2 and CDRL3 within SEQ ID NO: 1 (e.g. SEQ ID Nos.
  • SEQ ID NO:9 e.g., SEQ ID Nos: 10-12
  • SEQ ID NO: 17 e.g., SEQ ID NOs. 18-20
  • SEQ ID NO:25 e.g., SEQ ID Nos: 26-28
  • SEQ ID NO:33 e.g, SEQ ID Nos: 34-36
  • T cell or NK surface antigen antibodies or antigen binding fragments used in the bispecific antibodies are used in the bispecific antibodies.
  • the portion of the bispecific antibody that specifically and selectively binds to T cell or NK cell surface may be an antibody, e.g., monoclonal antibody or antigen binding fragment thereof.
  • T cell surface markers are known in the art and include, but are not limited to, for example, CD3.
  • the suitable anti-T cell surface antibodies are known in the art and include, but are not limited to, e.g., anti-CD3 antibody or antigen binding fragment thereof.
  • the anti-T cell surface antibody is an anti-CD3 antibody.
  • the anti-CD3 antibody to be used in the present invention may be any antibody as long as it is specific for CD3.
  • the CD3 antibody may be a CD3 monoclonal antibody, for example OKT3.
  • OKT3 a murine IgG2a mAb directed against the e-chain of the CD3 complex on human T lymphocytes (Salmeron et al, J. Immunol. 147 (1991), 3047-3052) and produced by a hybridoma with the ATCC deposit number of CRL 8001.
  • the NK cell surface antigen may by an NK surface antigen known in the art, for example, CD 16.
  • an NK surface antigen known in the art, for example, CD 16.
  • a bispecific antibody comprising an anti-CD30 antibody described herein and an anti-CD 16 antibody or antigen binding fragment thereof is contemplated.
  • polypeptide and nucleic acids described herein encompass those to which conservative modifications have been made.
  • conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site- directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are those in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the related art.
  • one or more amino acid residues within the antibody or antigen binding fragment thereof of the present invention may be replaced by other amino acid residues from the same side chain family, and the altered antibodies or antibody fragments thereof may be tested using the functional assays described herein.
  • conservative changes may even be made in the CDR region and not alter the functional binding of the antibody or antigen binding fragment thereof, which can be tested by the methods described herein.
  • BLAST Basic Local Alignment Search Tool
  • the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula (Karlin and Altschul, 1990), the disclosure of which is incorporated by reference in its entirety.
  • the BLAST programs can be used with the default parameters or with modified parameters provided by the user.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 85% sequence identity.
  • percent identity can be any integer from 75% to 100%. More preferred embodiments include at least: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
  • Substantial identity of amino acid sequences for purposes of this invention normally means polypeptide sequence identity of at least 85%.
  • Preferred percent identity of polypeptides can be any integer from 85% to 100%. More preferred embodiments include at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the bispecific antibody may wish to express as a fusion protein linking the anti-CD30 antibody to the anti-T cell surface antigen antibody (e.g., anti-CD3 antibody).
  • the bispecific antibody of the present invention with a protein linker between the two antibodies.
  • Standard molecular biology techniques e.g., restriction enzyme based sub-cloning, or homology based sub-cloning
  • the fusion protein is then produced as one peptide in a host cell (e.g., yeast, bacteria, insect, or mammalian cell) and purified before use.
  • a host cell e.g., yeast, bacteria, insect, or mammalian cell
  • the therapeutic does not need to be a whole protein.
  • it can be a single peptide chain comprising the VH and VL sequences of the CD30 antibody linked to the VH and VL sequences of the CD3 antibody.
  • the anti-CD30 and anti-T cell/NK cell antibody may be covalently linked as depicted in FIG. 14 and as described in "CD20-Targeted T Cells after Stem Cell Transplantation for High Risk and Refractory Non-Hodgkin's Lymphoma" Lum, Lawrence G. et al, Biology of Blood and Marrow Transplantation, Volume 19, Issue 6, 925 - 933, incorporated by reference in its entirety. Briefly, the process of heteroconjugation of anti- CD3 with anti-CD30 antibody is shown in FIG. 14. Figure 14A shows one embodiment of how the antibodies may be conjugated together, by the end of the Fc portions.
  • Figure 14B shows a model method of covalently linking the antibodies.
  • Step 1 shows cross-linking of Trauf s reagent to anti-CD3 (OKT3) mAb and the cross-linking of Sulpho-SMCC to anti-CD30;
  • step 2 shows the heteroconjugation of the cross-linked anti-CD3 with the cross-linked anti-CD30; and
  • step 3 shows formation of anti-CD3 - anti-CD20 BiAb (CD30Bi).
  • the bispecific antibodies of the invention can be produced by a process in which two immunoglobulin molecules are linked together using a cross linking agent such as N-succinimidyl 3-(2-pyridyldithiol)-propionate, S- acetylmercaptosuccinic acid anhydride or the like (J. Exp. Med., 163, 166 (1986)) or by a process in which Fab fragments of immunoglobulin molecules are linked together (Eur. J. Immunol., 19, 1437 (1989)).
  • a cross linking agent such as N-succinimidyl 3-(2-pyridyldithiol)-propionate, S- acetylmercaptosuccinic acid anhydride or the like
  • Fab fragments of immunoglobulin molecules are linked together
  • Methods of preparing bispecific antibodies of the present invention include those described in WO 2008119353 (Genmab), WO 2011131746 (Genmab) and reported by van der Neut- Kolfschoten et al. (Science. 2007 Sep 14;317(5844) : 1554-7).
  • Examples of other platforms useful for preparing bispecific antibodies include but are not limited to BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star) , Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab).
  • Another strategy to promote formation of heterodimers over homodimers is a "knob-into-hole” strategy in which a protuberance is introduced on a first heavy-chain polypeptide and a corresponding cavity in a second heavy- chain polypeptide, such that the protuberance can be positioned in the cavity at the interface of these two heavy chains so as to promote heterodimer formation and hinder homodimer formation.
  • protuberances are constructed by replacing small amino-acid side-chains from the interface of the first polypeptide with larger side chains.
  • Compensatory "cavities" of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino-acid side-chains with smaller ones (US patent 5,731, 168).
  • EP1870459 Choi
  • WO 2009089004 Amgen
  • EP1870459 Choi
  • WO 2009089004 Amgen
  • one or more residues that make up the CH3-CH3 interface in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable.
  • W02007110205 Merck
  • bispecific antibodies Another in vitro method for producing bispecific antibodies has been described in WO 2008119353 (Genmab), wherein a bispecific antibody is formed by "Fab-arm” or "half molecule” exchange (swapping of a heavy chain and attached light chain) between two monospecific IgG4- or IgG4-like antibodies upon incubation under reducing conditions.
  • the resulting product is a bispecific antibody having two Fab arms which may comprise different sequences.
  • nucleic acid that encodes for the bispecific antibodies or antigen binding fragment thereof described above.
  • Some embodiments provide an isolated polynucleotide encoding a bispecific antibody described herein.
  • term“nucleic acid” or“polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).
  • a recombinant expression cassette comprising a polynucleotide encoding the bispecific antibody or antigen binding fragment thereof of the present invention is also contemplated.
  • the polynucleotide may be under the control of a transcriptional promoter allowing the regulation of the transcription of said polynucleotide in a host cell.
  • Said polynucleotide can also be linked to appropriate control sequences allowing the regulation of its translation in a host cell.
  • the present invention also provides a expression vector or recombinant vector (e.g., a recombinant expression vector) comprising a polynucleotide encoding the bispefic antibodies according to the present invention.
  • said recombinant expression vector is a recombinant expression vector comprising an "expression cassette" or "expression construct" according to the present invention.
  • the polynucleotides may operatively linked to a transcriptional promoter (e.g., a heterologous promoter) allowing the construct to direct the transcription of said polynucleotide in a host cell.
  • a transcriptional promoter e.g., a heterologous promoter
  • vector mleudes a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the antibodies or fragments thereof.
  • viral vector Another type of vector, wherein additional DNA segments may be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced.
  • Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g. lentiviral vectors).
  • Vector includes expression vectors, such as viral vectors (e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (rAAV)), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (rAAV)
  • rAAV adeno-associated viruses
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the antibodies or fragments thereof.
  • vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced.
  • Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g. lentiviral vectors).
  • Vector includes expression vectors, such as viral vectors (e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (rAAV)), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (rAAV)
  • the present invention also provides a host cell able to express the bispecific antibody described herein.
  • the host cell is a fused hybridoma cell of two hydridoma cells in another embodiment, the host cell contains isolated nucleic acids or a recombinant expression cassette or a recombinant expression vector according to the present invention able to express the bispecific antibody.
  • the host cell can be a prokaryotic or eukaryotic host cell.
  • the host cell is capable of expressing the bispecific antibodies of the present invention. Suitable host cells include, but are not limited to, mammalian cells, bacterial cells and yeast cells. In some embodiments, the host cell may be a eukaryotic cell.
  • host ceil also includes a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants and "transformed cells which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, hut may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the invention includes a purified and isolated host cell comprising an expression vector containing an isolated nucleic acid capable of encoding the bispecific antibody.
  • the host cell can be any cell capable of expressing antibodies, for example fungi; mammalian cells; insect cells, using, for example, a baculovirus expression system; plant cells, such as, for example, com, rice, Arabidopsis, and the like. See, generally, Verma, R. et al., J Immunol Methods. 1998 Jul. 1; 216(1-2): 165-81.
  • the bispecific can be wholly or partially synthetically produced that are used to make the bispecific antibody.
  • the antibody may be from any appropriate source, for example recombinant sources and/or produced in transgenic animals or transgenic plants.
  • the bispecific antibody molecules can be produced in vitro or in vivo.
  • the anti-CD30 or anti-CD3 antibody or antibody fragment comprises at least the heavy chain variable region (VH) which generally comprises the antigen binding site.
  • the antibody or antibody fragment comprises the heavy chain variable region and light chain variable region (VL).
  • the bispecific antibody comprising the antibody or antibody fragment can be made that comprises all or a portion of a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgE, IgM or IgD constant region.
  • a heavy chain constant region such as an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgE, IgM or IgD constant region.
  • the antibody or antibody fragment can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region. All or part of such constant regions may be produced wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated bispecific antibody or antigen binding fragment thereof specific for human CD30 and a T cell surface antigen (e.g., CD3).
  • the composition further includes a suitable carrier, preferably a pharmaceutically acceptable carrier.
  • suitable carrier preferably a pharmaceutically acceptable carrier.
  • Compositions are provided that include one or more of the disclosed bispecific antibodies.
  • the compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome.
  • the bispecific antibody can be formulated for systemic or local (such as intravenous, intrathecal) administration.
  • “pharmaceutical composition” means therapeutically effective amounts of the antibody together with a pharmaceutically-acceptable carrier.
  • “Pharmaceutically acceptable” carriers are known in the art and include, but are not limited to, for example, suitable diluents, preservatives, solubilizers, emulsifiers, liposomes, nanoparticles and adjuvants.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01 to 0.1 M and preferably 0.05M phosphate buffer or 0.9% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include isotonic solutions, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • compositions of the present disclosure may include liquids or lyophilized or otherwise dried formulations and may include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.
  • solubilizing agents e.g., glycerol, polyethylene glycerol
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., Thimerosal, benzyl alcohol, parabens
  • bulking substances or tonicity modifiers e.g., lactose, mannitol
  • covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • Such compositions will influence the physical state, solubility, stability, rate of in vivo
  • the compositions comprise a pharmaceutically acceptable carrier, for example, buffered saline, and the like.
  • a pharmaceutically acceptable carrier for example, buffered saline, and the like.
  • the compositions can be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable additional substances as required to approximate physiological conditions such as a pH adjusting and buffering agent, toxicity adjusting agents, such as, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.
  • the antibodies are provided in lyophilized form and rehydrated with sterile water or saline solution before administration.
  • the antibodies are provided in sterile solution of known concentration.
  • the antibody composition may be added to an infusion bag containing 0.9% sodium chloride, USP and in some cases, administered in a dosage of from 0.5 to 15 mg/kg of body weight.
  • the bispecific antibodies described herein are able to stimulate a T cell or NK cell response to a specific CD30+ tumor cell.
  • the bispecific antibody bring T-cell via its CD3 receptor into close contact with the tumor cell (via its CD30 antigen) to form an immunological synapse, which generates a strong activating signal cell mediated signal.
  • the bispecific antibody allows the T-cells to be activated at the site of tumor cells allowing for a T-cell mediated response against the CD30+ tumor cell.
  • this activating signal induces the transport of cytotoxic granules to the cell surface where they release perforin and granzyme in the vicinity of the tumor cell.
  • granules containing cell lysing components such as perforin, granzyme and lysosomal enzymes, are transported towards the cell membrane of the T-cell. Once at the surface and subsequently secreted into the extracellular matrix. Perforin causes the formation of pores in the target cell, thereby facilitating the entry of the cell lysing components. This set of activities leads to tumor cell apoptosis.
  • the present disclosure provides a method of inhibiting or reducing growth of a tumor cell expressing CD30, comprising contacting the tumor cell with an effective amount of the bispecific antibody of such that the growth of the cell is inhibited.
  • inhibiting cell growth includes killing or apoptosising the cancer cell.
  • the method includes reducing, inhibiting or preventing growth of tumor cells.
  • the term "inhibits proliferation” (e.g. referring to cells, such as tumor cells) is intended to include any substantial decrease in the cell proliferation when contacted with a bispecific antibody as compared to the proliferation of the same cells not in contact with the bispecific antibody, e.g., the inhibition of proliferation of a cell culture by at least about 10%, at least about 20% or at least about 30%.
  • the antibodies of the invention specifically bind CD30 and T cells (via CD3) and exert cytostatic and cytotoxic effects on malignant cells in cancer (e.g., Hodgkin's lymphoma).
  • One embodiment of the present invention provides a method of treating a patient having a CD30 + cancer, the method comprising administering a therapeutically effective amount of the isolated bispecific antibody described herein capable of simultaneously binding human CD30 and T cell surface antigen as described herein to treat the cancer.
  • treating describes the management and care of a subject for the purpose of combating the disease, condition, or disorder. Treating includes the administration of an antibody of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.
  • the term "treating" can be characterized by one or more of the following: (a) the reducing, slowing or inhibiting the growth of cancer, including reducing slowing or inhibiting the growth of cancer cells; (b) preventing the further growth of tumors; (c) reducing or preventing the metastasis of cancer within a patient, and (d) reducing or ameliorating at least one symptom of the cancer.
  • the optimum effective amounts can be readily determined by one of ordinary skill in the art using routine experimentation.
  • the treatment can result in cell-cycle inhibition of tumor cells (i.e., cytostasis). In another embodiment, the treatment can result in a T-cell mediated killing of the tumor cells.
  • the terms“effective amount” and“therapeutically effective amount” refer to the quantity of active therapeutic agent or agents sufficient to yield a desired therapeutic response without undue adverse side effects such as toxicity, irritation, or allergic response.
  • the specific“effective amount” will, obviously, vary with such factors as the particular condition being treated, the physical condition of the subject, the type of animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
  • the bispecific antibody of the present invention is used for treatment in addition to standard treatment options, for example surgery and radiation therapy.
  • the antibodies of the present disclosure are used in combination therapy, e.g. therapy including one or more different anti-cancer agents.
  • Suitable CD30 + cancers include, hematologic malignancies, for example, Hodgkins lymphoma, anaplastic large cell lymphoma, acute myeloid leukemia (AML), ovarian cancer, mesothelioma, skin squamous cell carcinoma, triple negative breast cancer, pancreatic cancer, small cell lung cancer, anal cancer, and thyroid carcinoma, among others.
  • hematologic malignancies for example, Hodgkins lymphoma, anaplastic large cell lymphoma, acute myeloid leukemia (AML), ovarian cancer, mesothelioma, skin squamous cell carcinoma, triple negative breast cancer, pancreatic cancer, small cell lung cancer, anal cancer, and thyroid carcinoma, among others.
  • CD30 has also been shown to be expressed on a subset of non-Hodgkin's lymphomas (NHL), including Burkitfs lymphoma, anaplastic large-cell lymphomas (ALCL), cutaneous T-cell lymphomas, nodular small cleaved-cell lymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennerfs lymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), adult T-cell leukemia (T-ALL), and entroblastic/centrocytic (cb/cc) follicular lymphomas, along with embryonal carcinomas, nonembryonal carcinomas, malignant melanomas, and mesenchymal tumors.
  • the present methods may be used to treat any cancer in which CD30 + tumor cells are found.
  • administering refers to any method of providing a pharmaceutical preparation to a subject.
  • Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, intraaural administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, intrathecal administration and subcutaneous administration.
  • Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • the administration is intravenous administration.
  • the present invention also includes anti-CD3/anti-CD30 bispecific antibodies which exhibit one or more characteristics selected from the group consisting of: (a) inducing T cell proliferation; (b) activating T-cells, inducing IFN-gamma release; (c) inducing T-cell mediated cytotoxicity on anti-CD30+ tumor cells; (d) increasing CD30+ tumor cell death; and (e) decreasing CD30+ tumor volume.
  • kits for carrying out the methods described herein are provided.
  • the kits provided may contain the necessary components with which to carry out one or more of the above-noted methods.
  • kits for treating a subject with a CD30 + cancer are provided.
  • the kit comprises a bispecific antibody described herein and instructions for use. Further, the kit may comprise a pharmaceutically acceptable carrier and instructions for use.
  • transitional phrase“consisting essentially of’ limits the scope of a claim to the specified materials or steps“and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
  • Consisting of is a closed term that excludes any element, step or ingredient not specified in the claim.
  • sequences consisting of refers to the sequence listed in the SEQ ID NO. and does refer to larger sequences that may contain the SEQ ID as a portion thereof.
  • Example 1 Anti-CD30 monoclonal antibody production
  • FIG. 2 demonstrates binding of purified antibodies to cells with CD30 surface expression.
  • Microtiter plates were coated with recombinant CD30-GST fusion protein. Wells were blocked with 5% bovine serum albumin (BSA) solution. Purified 8D10, 10C2, 12B1, 13H1, 15B8, BY88 (commercial anti-CD30 antibody), AC10 (commercial anti-CD30 antibody) or MI 15 (commercial anti-CD 138 antibody) were added and incubated at varying concentrations. Wells were detected by incubating with an alkaline phosphatase-labeled anti mouse IgG antibody. The plate was developed with pNPP (p-nitrophenyl phosphate). The optical density at 405 was determined using a plate reader and the results are shown in FIG. 4.
  • BSA bovine serum albumin
  • CD30+ SU-DHL-l cells were blocked with unlabeled 8D10, 10C2, 12B1,
  • All five antibodies have unique light and heavy chain sequences. These sequences also differ from the FDA-approved anti-CD30 antibody AC 10 (Brentuximab vedotin). All five antibodies bind to CD30, as indicated by FACS and ELISA assays. All five antibodies bind to an epitope that is different from AC10 (Brentuximab). 8D10, 12B1, 13H1, 15B8 bind to the same or similar epitope as each other while 10C2 binds to an epitope that is distinct both from AC 10, and from the other four novel antibodies reported here.
  • FIG. 19 demonstrates the binding specificity of the new CD30 antibodies.
  • Example 2 Bispecific CD30-CD3 antibody as immunotherapy for Hodgkin Lymphoma
  • This Example demonstrates the ability to make bispecific antibodies that bind to CD30 and to T cells (via CD3) to produce an anti-cancer effect for Hodgkin's Lymphoma.
  • Two anti-CD30 clones (8D10 and 10C2) were covalently heteroconjugated to an anti-T cell antibody (CD3) (OKT3).
  • FACS was used to demonstrates that the hetero-conjugated CD30 antibodies bind to both a CD30+ Hodgkin's Lymphoma cell line (SU-DHL-l cells, FIG. 14B) and to primary human T cells (FIG. 14B) for both the 8Dl0biAb (red) and 10C2 biAb (blue).
  • the ability to stimulate the effector function of T cells was measured by production of pro-inflammatory cytokines IFN-g and IL-2.
  • 8D10 biAb or 10C2 biAb-armed T cells produce the pro-inflammatory cytokines IFN-g (A) and IL-2 (B) when co-cultured with CD30+ tumor cells, indicating that binding of T cells to the biAb is sufficient to trigger T cell activation and cytotoxicity.
  • Armed T cells cocultured with CD30- cells have much lower cytokine production demonstrating the specificity of this approach.
  • the cytotoxicity of the biAbs was also confirmed by cell death in the presence of the 8D10 and 10C2 biAb-armed T cells against CD30+ tumor cells.
  • CD30+ tumor cell lines SU-DHL-l, RPMI6666 and Raji LV30 are killed with varying efficacy by 8D10 and 10C2 biAb-armed T cells as demonstrated in FIG. 17.
  • Non- transduced Raji cells (FIG. 18A, CD30-) are not killed by the armed T cells.
  • FIG. 18B demonstrates that the CD30 expression on activated T cells is low enough to avoid elimination by the 8D10 and 10C2 biAb armed T cells.
  • this Example demonstrates the ability and specificity of the biAb to activate T cells and kill tumor cells.
  • the bispecific antibody results in the release of proinflammatory cytokines, which triggers T cell cytotoxicity specifically against CD30+ cells, while sparring CD30- cells. Also, T cell fratricide as a result of low CD30 expression on activated T cells is not observed.
  • DIVMTOSPASOSASLGESVTITCLASOTIGTWLAWYOOKPGKSPOFLIYAATSLADG VPSRF SGSGSGTKFSFKISSLOAEDFVSYYCOOLYSTPFTFGGGTKLEIK CDRL1- SEQ ID NO: 2-underline; CDRL2-SEQ ID NO:3- bold; CDRL3-SEQ ID NO: 4-bold/underline
  • DIVMTOTTASLSTSVGETVTITCRASGNLHSYLTWYOOKOGKSPOLLVYNAKTLAD GVPSRFSGSGSGTOYSLKIDSLOPEDFGSYYCOHFWTTPFTFGSGTKLEIK CDRL1- SEQ ID NO: 18-underline; CDRL2-SEQ ID NO: 19- bold; CDRL3-SEQ ID NO:20- bold/underline
  • DIVMTOTPKSMSMSVGERVTLSCKASENVGTYVSWYOOKPEOSPKVLIYGASNRFT GVPDRFTGSGSATDFTLTISSVOTEDLADYHCGOSYSYPLTFGAGTKLELK (CDRL1- SEQ ID NO: 26-underline; CDRL2-SEQ ID NO:27- bold; CDRL3-SEQ ID NO:28- bold/underline) [00151] 13H1 -Heavy chain (SEQ ID NO: 29)
  • DIVMTOTPASLSASVGETVTITCRASGNIHNYLAWYOOKOGKSPOLLVYNAKTLAD GVPSRF SGSGSGTOYSLKINSLOPEDFGSYYCOHFWSTPFTFGSGTKLEIK CDRL1- SEQ ID NO: 34-underline; CDRL2-SEQ ID NO:35- bold; CDRL3-SEQ ID NO:36- bold/underline

Abstract

La présente invention concerne de nouveaux anticorps bispécifiques qui se lient à CD30 humain et leurs utilisations. L'invention concerne également des méthodes de traitement du cancer utilisant les anticorps bispécifiques fournis par l'invention.
EP19783844.4A 2018-09-24 2019-09-24 Système et procédé de développement d'anticorps bispécifiques cd30 pour l'immunothérapie de malignités cd30+ Pending EP3856778A1 (fr)

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CA3155291A1 (fr) * 2018-09-24 2020-04-02 The Medical College Of Wisconsin, Inc. Anticorps anti-cd30 et procedes d'utilisation
AU2022231068A1 (en) 2021-03-01 2023-08-17 Nantbio, Inc. Anti-cd30 monoclonal antibodies and chimeric antigen receptors
TW202330610A (zh) 2021-10-08 2023-08-01 丹麥商珍美寶股份有限公司 結合至ｃｄ３０及ｃｄ３之抗體

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US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
NZ534174A (en) * 2002-01-09 2007-03-30 Medarex Inc An isolated human monoclonal antibody which binds to human CD30
ES2592271T3 (es) 2005-03-31 2016-11-29 Chugai Seiyaku Kabushiki Kaisha Métodos de producción de polipéptidos mediante la regulación de la asociación de los polipéptidos
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